Does heterochromatin protein 1 always follow code?

Heterochromatin protein 1 (HP1) is a conserved chromosomal protein that participates in chromatin packaging and gene silencing. A loss of HP1 leads to lethality in Drosophila and correlates with metastasis in human breast cancer cells. On Drosophila polytene chromosomes HP1 is localized to centric regions, telomeric regions, in a banded pattern along the fourth chromosome, and at many sites scattered throughout the euchromatic arms. Recently, one mechanism of HP1 chromosome association was revealed; the amino-terminal chromo domain of HP1 interacts with methylated lysine nine of histone H3, consistent with the histone code hypothesis. Compelling data support this mechanism of HP1 association at centric regions. Is this the only mechanism by which HP1 associates with chromosomes? Interest is now shifting toward the role of HP1 within euchromatic domains. Accumulating evidence in Drosophila and mammals suggests that HP1 associates with chromosomes through interactions with nonhistone chromosomal proteins at locations other than centric heterochromatin. Does HP1 play a similar role in chromatin packaging and gene regulation at these sites as it does in centric heterochromatin? Does HP1 associate with the same proteins at these sites as it does in centric heterochromatin? A first step toward answering these questions is the identification of sequences associated with HP1 within euchromatic domains. Such sequences are likely to include HP1 "target genes" whose discovery will aid in our understanding of HP1 lethality in Drosophila and metastasis of breast cancer cells.     

Heterochromatic deposition of centromeric histone H3-like proteins

Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. The functional significance of this centromere-heterochromatin relationship, if any, is unknown. One possibility is that heterochromatin provides a suitable environment for assembly of centromere components, such as special centromeric nucleosomes that contain distinctive histone H3-like proteins. We describe a Drosophila H3-like protein, Cid (for centromere identifier) that localizes exclusively to fly centromeres. When the cid upstream region drives expression of H3 and H2B histone-green fluorescent protein fusion genes in Drosophila cells, euchromatin-specific deposition results. Remarkably, when the cid upstream region drives expression of yeast, worm, and human centromeric histone-green fluorescent protein fusion proteins, localization is preferentially within Drosophila pericentric heterochromatin. Heterochromatin-specific localization also was seen for yeast and worm centromeric proteins constitutively expressed in human cells. Preferential localization to heterochromatin in heterologous systems is unexpected if centromere-specific or site-specific factors determine H3-like protein localization to centromeres. Rather, the heterochromatic state itself may help localize centromeric components.     

RNAi and heterochromatin repress centromeric meiotic recombination

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.     

Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

The origin recognition complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) α- and HP1β-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subcomplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1α, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1α; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1α association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1α distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1α from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1α-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function.     

Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1

The polytene chromosomes of Drosophila melanogaster consist of condensed heterochromatin regions most of which are in the chromocenter, telomeres, and the fourth chromosome. Whereas suppressor of variegation 3-9 [SU(VAR)3-9], a histone methyltransferase, is mainly responsible for lysine 9 of histone H3 (H3K9) methylation of the chromocenter and consequent binding of the heterochromatin-protein HP1, the enzyme for painting of the fourth chromosome by H3K9 methylation has been elusive. We show here that dSETDB1, the Drosophila ortholog of the mammalian SETDB1, is an authentic H3K9 methyltransferase and a pleiotropic regulator of the fly's development. Drosophila mutants hypomorphic or null in dSETDB1 expression lose most of the H3K9 methylation as well as HP1-binding on the fourth chromosome. We also show that binding of painting of fourth (POF), a known fourth chromosome-specific protein, and the dSETDB1-controlled H3K9 methylation of this chromosome are interdependent. Furthermore, POF and dSETDB1 interact with each other in vivo. The deregulation of H3K9 methylation, HP1-binding, and POF-binding resulted in, on the average, a global reduction of gene expression from the fourth chromosome but not the other chromosomes. Deficiency of dSETDB1 also up-regulated the expression of HP1. These results have suggested an interactive network, as controlled in part by dSETDB1, regulating the epigenetic modification and gene expression of Drosophila chromosome 4.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Histone H3 N-terminus regulates higher order structure of yeast heterochromatin

In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.     

Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA.DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The chromosome 3 locus is the only band in the polytene genome that shows variation in size and intensity of staining between populations and species. The interband chromosome regions that are immediately distal or proximal to the 18/28S rRNA locus have been involved in a disproportionately large number of natural inversion breaks observed in the euchromatic portion of the polytene chromosome. In 104 species of Hawaiian Drosophila in which chromosome 3 polytene sequences have been determined, 15 breaks occur in these two regions. On a random basis, only one such break is expected. We propose that this locus may be flanked by substantial heterochromatic blocks which are not represented in the salivary gland chromosome.     

Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood

OBJECTIVE: During terminal cell differentiation, nuclear chromatin becomes condensed and the repertoire of epigentic heterochromatin proteins responsible for chromatin condensation is dramatically changed. In order to identify the chromatin regulatory factors associated with incomplete cell differentiation and impaired chromatin condensation in hematological malignancies, we examined expression levels of major heterochromatin proteins in normal blood cells and cells derived from a number of chronic and acute myeloid leukemia patients exhibiting different degrees of differentiation. METHODS: We used immunoblotting and immunofluorescence to examine the levels and localization of epigenetic heterochromatin factors in isolated cell nuclei and fractionated peripheral blood cells. RESULTS: While the major epigenetic heterochromatin factor, histone H3 methylated at lysine 9, is present in all cell types, its main counterparts, nonhistone proteins, heterochromatin proteins 1 (HP1) alpha, beta, and gamma, are dramatically reduced in peripheral blood leukocytes of normal donors and chronic myeloid leukemia patients, but are substantially increased in the blood of accelerated phase and blast crisis patients. In the terminally differentiated cells, nuclear chromatin accumulates a nucleocytoplasmic serpin, monocyte and neutrophil elastase inhibitor (MNEI). HP1 and MNEI levels inversely correlate in a number of normal and leukemia myeloid cells and show strikingly opposite coordinated changes during differentiation of U937 cell line induced by retinoic acid. CONCLUSIONS: Our results suggest that repression of HP1 and accumulation of MNEI are linked to terminal cell differentiation and that their levels may be monitored in blood cell populations to detect transitions in cell differentiation associated with leukemia progression and treatment.     

H2A.Z contributes to the unique 3D structure of the centromere

Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure.